Table I. Phenotypes of mutations analyzed in this studya.
| Alleleb | Mutationc | Binding domain affectedd | Co-purifying factorse | Complementing functionf | Dom. Slg–g/suppressed byh | Dom. Gcd–i/suppressed by hc eIF2-IMT4j |
|---|---|---|---|---|---|---|
| TIF32 mutations | ||||||
| lc empty vector | NA | NA | None | – | – | – |
| hc TIF32-His | None | None | All | + | – | – |
| lc TIF32-Δ8-His | [Δ1–199] | None | All | Partial | – | ND |
| hc TIF32-Δ8-His | [Δ1–199] | None | ND | Partial | + | – |
| lc TIF32-Δ86-His | [Δ1–199; 791–964] | PRT1k, HCR1, eIF2 | All | – | – | ND |
| hc TIF32-Δ86-His | [Δ1–199; 791–964] | PRT1k, HCR1, eIF2 | ND | – | – | – |
| hc TIF32-Δ4-His | [Δ1–790] | PRT1k, NIP1, eIF1 | eIF2 | – | – | – |
| lc TIF32-Δ5-His | [Δ642–964] | PRT1, HCR1, eIFs 1, 2 | NIP1, eIF5 | – | – | ND |
| hc TIF32-Δ5- His | [Δ642–964] | PRT1, HCR1, eIFs 1, 2 | ND | – | +/hc NIP1 | – |
| +/hc eIF2 + IMT4 (partial) | ||||||
| lc TIF32-Δ6-His | [Δ791–964] | PRT1k, HCR1, eIF2 | NIP1, PRT1, TIF34, TIF35, eIFs 1, 5 | – | – | ND |
| hc TIF32-Δ6-His | [Δ791–964] | PRT1k, HCR1, eIF2 | ND | – | +/hc eIF2 + IMT4 | – |
| |
||||||
| NIP1 mutations | ||||||
| sc empty vector | NA | NA | None | – | – | – |
| hc NIP1-His | None | None | All | + | – | –/ND |
| sc NIP1-N′-His | [Δ206–812] | TIF32, PRT1 | eIFs 1, 2, 5 | – | – | – |
| hc NIP1-N′-His | [Δ206–812] | TIF32, PRT1 | eIFs 1, 2, 5 | – | – | ++/Yes |
| sc NIP1-C-His | [Δ1–156] | eIF1, eIF5 | PRT1, TIFs 32, 34, 35 | – | – | – |
| hc NIP1-C-His | [Δ1–156] | eIF1, eIF5 | PRT1, TIFs 32, 34, 35 | – | – | – |
| hc NIP1-ΔJ-His | [Δ157–371] | TIF32 | eIFs 1, 2, 5 | – | – | – |
| hc NIP1-ΔA-His | [Δ371–812] | PRT1 | TIF32, eIFs 1, 2, 5 | – | – | – |
| hc NIP1-ΔB′-His | [Δ571–812] | PRT1 | All | – | – | – |
| |
||||||
| PRT1 mutations | ||||||
| sc empty vector | NA | NA | None | – | – | – |
| hc PRT1-His | None | None | All | + | – | – |
| hc PRT1-Δ5′-His | [Δ551–724] | NIP1, TIFs 34, 35 | TIF32 | – | – | – |
| sc PRT1-Δ7′-His | [Δ641–724] | TIFs 34, 35 | TIF32, NIP1, eIF5 | – | – | ND |
| hc PRT1-Δ7′-His | [Δ641–724] | TIFs 34, 35 | ND | – | +/hc NIP1 + TIF32 | – |
| sc PRT1-ΔRRM-Hisl | [Δ35–136] | TIF32, HCR1 | TIFs 34, 35 | – | +/hc TIF34 + TIF35 | ND |
aThe LEU2 or URA3 plasmids containing TIF32-His, NIP1-His, PRT1-His, or their mutant derivatives, were introduced into strains YLV314U [MATa ura3::URA3::rpg1-1 trp1-1::TRP1::rpg1-Δ2 ade2-1 can1-100 leu2-3 112 his3-11,15], B8302 [MATa cyc1-NLS cyc7-67 ura3-52 lys5-10 nip1-1] or H1676 [MATa prt1-1 leu2-3 leu2-112 ura3-52], respectively, to analyze complementation of the Ts– phenotypes of these strains. All of the plasmids were also introduced into W303 [MATa ade2-1 trp1-1 can1-100 leu2-3 112 his3-11,15 ura3] and H2881 [MATa trp1-1 leu2-3 112 ura3-52 gcn2Δ] to test for dominant slow growth (Slg–) or Gcd– phenotypes, respectively. NA, not applicable; ND, not determined.
bSingle-copy (sc) vectors YCplac33 for NIP1 and YCplac11 for PRT1; low-copy (lc) vector pRS315 for TIF32; high-copy (hc) vector YEplac181 for TIF32, NIP1 and PRT1.
cNumbers in parentheses indicate the amino acids of the particular protein that were deleted.
dComplete or partial deletion of the predicted binding domain(s) for the listed protein(s) in the His8-tagged protein under study.
eList of protein(s) that co-purified with the His8-tagged protein under study in Ni2+ chelation chromatography, summarized from Figures 2–4.
fComplementation of the growth defect at 37°C in transformants of the rpg1-1, nip1-1 or prt1-1 Ts– strains YLV314U, B8302 and H1676, respectively, after 3 days of incubation; –, no complementation; +, complementation similar to the wild-type allele.
gDom. Slg–, dominant slow growth phenotype at 30°C in the presence of a wild-type chromosomal copy of the corresponding gene in W303; –, no dominant Slg– phenotype; +, dominant Slg– phenotype evident.
hPlasmids YEpNIP1T carrying NIP1; p1780-IMT containing SUI2, SUI3, SUI4 and IMT4 (encoding the three subunits of eIF2 and tRNAiMet, respectively), and pLPY-NIP1-TIF32 bearing NIP1 and TIF32 were used to complement the dominant Slg– phenotype conferred by the relevant mutant alleles in W303 at 30°C.
iGcd– phenotypes were recognized by suppression of the 3-AT-sensitive phenotype of the gcn2Δ allele in transformants of H2881 evaluated on SC-Leu-His medium containing 10 mM 3-AT for the TIF32-His and PRT1-His alleles and on SC-Leu-His with 20 mM 3-AT for the NIP1-His alleles. Growth of isogenic GCN2 strain H2880 [MATa trp1-1 leu2-3,112 ura3-52] and the gcn2Δ strain H2881 transformed with an empty vector on these media were scored as +++++ and –, respectively.
jStrains containing particular NIP1-His alleles were transformed with p1780-IMT and tested for suppression of the Gcd– phenotype under the same conditions as in i, except that SC-Leu-Ura-His with 20 mM 3-AT was used; Yes, complete suppression; No, no suppression.
kOnly one of the two PRT1-binding domains in TIF32 affected.
lDetermined in Valášek et al. (2001).